PET Imaging of Small Extracellular Vesicles via [89Zr]Zr(oxinate)4 Direct Radiolabeling

Exosomes or small extracellular vesicles (sEVs) are increasingly gaining attention for their potential as drug delivery systems and biomarkers of disease. Therefore, it is important to understand their in vivo biodistribution using imaging techniques that allow tracking over time and at the whole-body level. Positron emission tomography (PET) allows short- and long-term whole-body tracking of radiolabeled compounds in both animals and humans and with excellent quantification properties compared to other nuclear imaging techniques. In this report, we explored the use of [89Zr]Zr(oxinate)4 (a cell and liposome radiotracer) for direct and intraluminal radiolabeling of several types of sEVs, achieving high radiolabeling yields. The radiosynthesis and radiolabeling protocols were optimized for sEV labeling, avoiding sEV damage, as demonstrated using several characterizations (cryoEM, nanoparticle tracking analysis, dot blot, and flow cytometry) and in vitro techniques. Using pancreatic cancer sEVs (PANC1) in a healthy mouse model, we showed that it is possible to track 89Zr-labeled sEVs in vivo using PET imaging for at least up to 24 h. We also report differential biodistribution of intact sEVs compared to intentionally heat-damaged sEVs, with significantly reduced spleen uptake for the latter. Therefore, we conclude that 89Zr-labeled sEVs using this method can reliably be used for in vivo PET tracking and thus allow efficient exploration of their potential as drug delivery systems.


■ INTRODUCTION
Exosomes, better described as small extracellular vesicles (sEVs), are cell-derived nanovesicles enclosed by a phospholipid bilayer, secreted by most cell types. 1 They are formed inside endosomal multivesicular bodies and released into the extracellular space by exocytosis. sEVs are small in size (30− 150 nm) and characterized by the presence of specific membrane-marker proteins such as CD63, CD9, Alix, and TSG101. 2 The role of sEVs is the transport and exchange of cytosolic molecules (i.e., nucleic acids, lipids, proteins, etc.) between cells, 3 thus acting as messengers in cell−cell communication and disease progression. 4 For example, tumor cell sEVs have been shown to promote tumor cell proliferation 5 and metastasis 6 and induce anticancer drug resistance. 7 Interestingly, natural and drug-loaded sEVs (derived from stem cells, immune cells, or cancer cells) have shown therapeutic potential in cancer, 8 Alzheimer's disease, 9 and type 2 diabetes. 10 Furthermore, they have the ability to cross the blood−brain barrier (BBB) 11 and to selectively target tissues. 12 Therefore, there is an increasing interest in the use of sEVs as nanotherapeutics. 13 In this context, it is important to develop imaging tools that track the in vivo behavior of sEVs.
Doing so will improve our understanding of their biology and also support their development as drug delivery tools.
Optical imaging has been used to investigate the distribution of sEVs, 14 but with associated challenges in quantification and signal tissue penetration. Radionuclide imaging can overcome these limitations. In particular, positron emission tomography (PET) imaging allows sensitive and quantitative whole-body imaging, with no background signal and unlimited tissue penetration in both animals and humans. 15 At the time of writing, there are only a handful of peer-reviewed publications on the radiolabeling and in vivo imaging of sEVs, 16−30 of which only three were aimed for PET imaging using three different radionuclides ( 64 Cu, 68 Ga, and 124 I). 24−27 These PET radiolabeling methods rely on the binding of radionuclides to membrane proteins which, given the importance of these surface components in the role of sEVs as messengers and cell−cell communication, may result in altered biodistribution and function as previously shown with 111 In-and 124 I-labeled sEVs. 16,26 Consequently, radiolabeling within the intraluminal space of sEVs is desirable.
Based on our previous work on cell and liposome radiolabeling, 31−33 we hypothesized that radiometal complexes that are metastable, lipophilic, and neutral, such as those based on ionophore ligands, would allow intraluminal sEV radiolabeling (Scheme 1). In particular, the PET radionuclide 89 Zr complexed by 8-hydroxyquinoline (oxine) allows direct radiolabeling of liposomes demonstrating intraluminal delivery of 89 Zr across the lipid bilayer of vesicles. 31 Here, we report a radiochemical synthesis method of [ 89 Zr]Zr(oxinate) 4 that allows efficient radiolabeling of sEVs and in vivo tracking using PET imaging.
The lipophilic [ 89 Zr]Zr(oxinate) 4 complex is able to pass through the lipid bilayer of the vesicles where 89 Zr dissociates from the oxine ligands (that presumably become protonated and are able to cross the lipid bilayer), and 89 Zr binds to intravesicular metal chelating ligands, such as proteins and nucleic acids, within the sEV.  4 synthesis was optimized for sEV radiolabeling ( Figure 1A). In particular, the final solution had to be isosmotic to avoid sEV damage and with a high 89 Zr concentration for in vivo PET studies. To achieve this, our synthesis involved the conversion of [ 89 Zr]Zr(oxalate) 4 in 1 M oxalic acid, as received from cyclotron production, into [ 89 Zr]ZrCl 4 (in 1 M HCl) by ion exchange chromatography. 34 This was followed by a drying step involving gentle heating under a flow of N 2 gas to remove HCl and H 2 O and allowing the concentration of the radioactivity. At this point, 80 μL of the oxine kit containing 1 M HEPES, 40 μg (0.3 μmol) of oxine, and 1 mg/mL polysorbate-80 at pH 7.8 was added (Method 1). 35 4 was also synthesized using an alternative method (Method 2) involving reaction of [ 89 Zr]ZrCl 4 with oxine as a solution in EtOH, followed by pH neutralization. No significant differences were observed between the two methods, based on RCY and logD 7.4 assessments ( Figure S1B). However, radiolabeling of sEVs using Method 1 was found to be highly reproducible and stable, hence was chosen for in vivo PET imaging studies.
Isolation and Characterization of sEVs. As the release of sEVs from cancer cells is considerably higher than from normal cells, 36 Figure 2A). To determine the purity of the isolated sEVs, the particle-toprotein (P:P) ratio was measured. This ratio developed by Webber and Clayton 39 determines the level of protein contamination in sEV samples, and a ratio < 1.5 × 10 9 is considered "unpure". A P:P ratio of > 1 × 10 10 sEVs/μg protein was achieved for both MDA-MB-231.CD63-GFP and PANC1 sEVs ( Figure 2B), indicating the purity of the isolated sEVs. Dot blot analysis of both sEVs demonstrated presence of sEV membrane markers CD63, CD81, and CD9, although CD81 was not detected on the MDA-MB-231.CD63-GFP sEVs. Furthermore, presence of Alix (endosomal protein) and absence of calnexin (endoplasmic reticulum-associated protein) indicated the endosomal origin (i.e., definition of exosomes) and purity of the isolated sEVs ( Figure 2C).
Radiolabeling of sEVs with [ 89 Zr]Zr(oxinate) 4 . We then tested the sEV radiolabeling capabilities of [ 89 Zr]Zr(oxinate) 4 . sEVs were incubated with [ 89 Zr]Zr(oxinate) 4 for 20 min at 37°C ( Figure 3A). These conditions were chosen based on our previous studies showing that [ 89 Zr]Zr(oxinate) 4 cell radio-labeling is temperature-independent and rapid (<20 min). 32 Following incubation, a small amount of the Zr chelator, desferrioxamine (DFO), was added to scavenge free 89 Zr 4+ ions from the reaction, including those that may be associated to the phospholipid membrane, as previously observed with liposomal vesicles. 40 This ensures that 89 Zr is only incorporated in the inside of the vesicles, by allowing efficient removal of any free or weakly bound extravesicular 89 Zr via size exclusion chromatography (SEC). The same sEV radiolabeling procedure was performed using non-chelated 89 Zr as a control ( 89 Zr-control)the same synthesis protocol and formulation as those of [ 89 Zr]Zr(oxinate) 4 but lacking oxine. The reaction mixture was then purified by Sepharose-based SEC systems that effectively separated sEVs from smaller molecules, including DFO-bound 89 Zr ( Figure S2). The results demonstrated significantly higher radiolabeling yields (RLYs) with [ 89 Zr]Zr(oxinate) 4 compared to 89 Zr-control for both sEVs ( Figure 3B), supporting our hypothesized radiolabeling strategy. Thus, [ 89 Zr]Zr(oxinate) 4 and not unchelated 89 Zris able to pass through the lipid bilayer membrane into the intraluminal space of sEVs where 89 Zr exchanges ligands and binds to intravesicular metal-chelating components, as we have previously demonstrated in cells and liposomes. 31−33 Furthermore, the addition of DFO did not Bioconjugate Chemistry pubs.acs.org/bc Article have any significant effect on sEV radiolabeling, suggesting that DFO neither enhances nor hinders the process ( Figure S3A). Tween-80, a common surfactant, is also present in the [ 89 Zr]Zr(oxinate) 4 formulation at a concentration of 1 mg/mL. The concentration of Tween-80 per radiolabeling reaction is ∼0.04 mg/mL, which is higher than its critical micellar concentration (0.02 mg/mL). 41 Whereas this reagent is important to provide long-term in vitro stability to [ 89 Zr]Zr-(oxinate) 4 , 35 it raises the concern that potential encapsulation of 89 Zr by Tween-80 micelles may be involved in the sEV radiolabeling process. To exclude this possibility, we performed an experiment whereby an equal number of PANC1 sEVs were radiolabeled with [ 89 Zr]Zr(oxinate) 4 and their corresponding oxine-free 89 Zr-control formulations, using both Methods 1 (containing Tween) and 2 (lacking Tween). The results showed that the presence of Tween-80 does not affect the RLYs of sEVs and hence that Tween is not involved in the radiolabeling reaction ( Figure S3B).
There was no significant change in the hydrodynamic size of PANC1 sEVs before and after radiolabeling (p = 0.4754, n = 4), unlike MDA-MB-231.CD63-GFP sEVs (p = 0.0138, n = 4− 8; Figure 3C). Despite detecting the sEV marker proteins CD63 and CD9 in both sEVs before and after radiolabeling ( Figure 3D), the size instability of MDA-MB-231.CD63-GFP sEVs after radiolabeling prompted us to select PANC1 sEVs for further in vitro and in vivo experiments. There were no Bioconjugate Chemistry pubs.acs.org/bc Article changes in the morphology of PANC1 sEVs, as analyzed by cryo-electron microscopy (cryoEM) ( Figure 3E). Additionally, flow cytometry analysis of PANC1 sEVs' membrane markers CD9, CD63, and CD81 pre-and post-radiolabeling further supports our hypothesis that intraluminal radiolabeling does not affect these membrane proteins ( Figure 3F). This conclusion was reached because flow cytometry requires conjugation of beads to the sEVs, and thus their detection relies on intact vesicles (vide inf ra). However, further studies, such as proteomics, will be required to validate this. In vitro radiochemical stability was analyzed by instant thin-layer chromatography (iTLC) using 10 mM EDTA as the mobile phase to detect 89 Zr 4+ ions released from the vesicles, showing that 89 Zr-PANC1 sEVs were 75.7 ± 3.4% (n = 3) stable after 26 h in phosphate-buffered saline (PBS) ( Figure S4).
In Vitro Cell Uptake of 89 Zr-Labeled PANC1 sEVs. Next, the ability of 89 Zr-PANC1 sEVs to be taken up by different types of cells in serum-supplemented media was evaluated. The 89 Zr-PANC1 sEVs, [ 89 Zr]Zr(oxinate) 4 , and 89 Zr-control were incubated at 37°C with the following cells: PANC1 (parental cells), HEK-293T (healthy cells with known nanoparticle-uptake properties), 42 MDA-MB-231, and DU-145 (non-parental cancer cells). Interestingly, 89 Zr uptake by both PANC1 cells and HEK-293T cells was significantly higher for the 89 Zr-PANC1 sEV group, compared to the two control groups ( Figure 4A,B). In contrast, there were very low levels of 89 Zr-PANC1-sEV uptake by the non-parental cancer cell lines ( Figure 4C,D). It is worth highlighting the higher uptake of 89 Zr-PANC1 sEVs in both PANC1 and HEK-293T cells compared to that achieved by [ 89 Zr]Zr(oxinate) 4 , taking into account that the latter has proven cell-radiolabeling properties. 32 Thus, these data demonstrate quick uptake of 89 Zr-

PANC1 sEVs by both parental cells and HEK-293T cells but not by other non-parental cancer cells.
In Vivo PET-CT Imaging of 89 Zr-PANC1 sEVs. Encouraged by these results, we performed an in vivo PET-CT imaging and biodistribution study of PANC1 sEVs in healthy mice (C57BL/6). Immunocompetent healthy mice, and not diseased animals, were chosen as the best model to test our radiolabeling approach, as they provide a baseline for future applications of this radiolabeling methodology and allow direct comparison with other methods. Based on the in vitro stability studies ( Figure S4), in vivo PET imaging was limited to 24 h, to minimize image/biodistribution analysis errors due to released free 89 Zr. To assess the impact of damaged vesicles on the imaging of sEVs, we evaluated three groups: (i) intact 89 Zr-PANC1 sEVs, (ii) heat-damaged 89 Zr-PANC1 sEVs, and (iii) neutralized 89 ZrCl 4 ( 89 Zr 4+ ). The heat-damage protocol consisted of two cycles of heating and cooling (90°C to 0°C) 89 Zr-PANC1 sEVs and was aimed at denaturing the vesicles but avoiding complete breakdown. Indeed, the heat-damage process resulted in an increase in size and partial release of internal contents ( Figure S5A) and damage of sEV surface marker proteins compared to intact 89 Zr-PANC1 sEVs ( Figure  S5B). 89 Zr-PANC1 sEVs were prepared with a RLY of 32% (1 × 10 12 sEVs). PET-CT imaging within 1 h post intravenous (iv.) injection (∼1 × 10 10 sEVs/mouse) showed short circulation times and rapid uptake of intact 89 Zr-PANC1 sEVs in the liver, spleen, bladder, several lymph nodes (LNs) [ Figure 5A(i)], and brain [ Figure 5B].
Short circulation times and liver/spleen/bladder uptake have been observed in other imaging studies of sEV biodistribution via iv. administration. [16][17][18]22,24,25 However, to the best of our knowledge, this is the first time LN uptake is observed using in vivo imaging. With the help of CT imaging, the PET signals Bioconjugate Chemistry pubs.acs.org/bc Article observed from the suspected LNs can be correlated with their well-documented location in mice (e.g., cervical, brachial, pancreatic, renal, inguinal, popliteal, and others; Figure S6). sEV/exosome uptake in secondary lymphoid organs (i.e., spleen and LNs) following iv. injection in the same mouse strain has been demonstrated and is mediated by CD169 + macrophages. 43 Interestingly, sEVs are known to express α-2,3linked sialic acid, which is the preferred ligand of CD169 thus providing a plausible explanation for the high spleen/LN uptake observed. 44 It should be noted that not all mice showed clear LN uptake and hence was not possible to identify them and isolate them ex vivo for further analysis. The possibility of these imaging signals being due to released free 89 Zr seems improbable due to its significantly different biodistribution [ Figure 5A(iii),C]. In addition, intact 89 Zr-PANC1 sEVs were visible within the brain ( Figure 5B) but not in the heatdamaged 89 Zr-PANC1 sEV group ( Figure S7), supporting the previously reported ability of sEVs to cross the BBB. 11 Heatdamaged 89 Zr-PANC1 sEVs showed a similar biodistribution to intact 89 Zr-PANC1 sEVs, with the major differences being a significantly lower spleen uptake and a higher bone signal [ Figure 5A(ii)]. These two findings can be explained by the Bioconjugate Chemistry pubs.acs.org/bc Article bigger size of the denatured vesicles and the partial release of contents we observed in vitro (vide supra), as a result of the heat-damaging process. In both groups, the bone signal increased at 24 h postinjection. This was expected and presumably due to the metabolic activity in the liver/spleen that will result in the release of bone-tropic "free" 89 Zr. In addition, fewer LNs were visible, and no brain signal was observed.
The PET-CT imaging findings correlated with the ex vivo biodistribution data. Comparison of the intact 89 Zr-PANC1 sEVs between 2.5 and 24 h suggests that once sEVs were taken up by the liver and the spleen, 89 Zr remained in these organs, as no difference was observed in the liver and spleen signal between the two time points (Figure S8A). At 24 h post injection, a high liver/spleen signal and higher uptake of intact 89 Zr-PANC1 sEVs in the spleen (55.7 ± 10.2 %ID/g) were observed, compared to heat-damaged 89 Zr-PANC1 sEVs (20.1 ± 7.5 %ID/g), p = 0.0040 ( Figure 5C and Table S1). The liver uptake was also higher for intact 89 Zr-PANC1 sEVs, whereas the bone uptake was higher for heat-damaged 89 Zr-PANC1 Figure 6. Ex vivo immunofluorescence detection of 89 Zr-PANC1 sEVs. (A) Spleen, (B) liver, and (C) kidney sections from mice injected with no sEVs (control), intact 89 Zr-PANC1 sEVs, and heat-damaged 89 Zr-PANC1 sEVs were stained with anti-human CD63-Cy5 (red) and DAPI (blue) for cell nuclei. All samples were obtained, stained, and imaged using the same conditions/settings. Scale bar = 50 μm. (D) Random ROIs were drawn on the Cy5 images, and the signal intensity was calculated using ImageJ; data presented as mean ± SD of n = 3 and analyzed using one-way ANOVA. Zr released from the vesicles accumulates in the bone, as evident by the increased bone uptake from 3.6 ± 0.8 %ID/g at 2.5 h to 7.2 ± 1.3 %ID/g at 24 h (p = 0.0015, unpaired t-test; Figure S8A). This was also confirmed by the higher liver:bone and spleen:bone uptake ratio at 2.5 h (Figure S8B), compared to 24 h (Figures 5D and S8C). A differential uptake of intact versus heat-damaged 89 Zr-PANC1 sEVs was observed for the spleen:bone uptake ratio (8.1 ± 2.6 vs 2.5 ± 1.7, respectively), suggesting a potential role of this ratio as an imaging biomarker for assessing the in vivo radiochemical stability of sEVs radiolabeled using this method.
Ex Vivo Immunofluorescence Detection of PANC1 sEVs. To confirm that the 89 Zr detected in the in vivo imaging and ex vivo biodistribution is from 89 Zr-labeled PANC1 sEVs, immunofluorescence detection of some key organs was performed. Thus, the spleen, liver (highest sEV uptake), and kidney (very low sEV uptake) were probed for anti-human CD63-Cy5 to detect PANC1 sEVs ( Figure 6). Tissues from C57BL/6j mice that had not been injected with sEVs served as the control for background fluorescence. Brighter fluorescence was observed in the spleen injected with intact PANC1 sEVs compared to heat-damaged sEVs, correlating with the PET imaging and ex vivo biodistribution data ( Figure 6A). A similar finding was observed in the liver ( Figure 6B), with increased presence of human CD63 in the intact sEV group, although the higher signal from the PET/ex vivo biodistribution experiments in this organ was not statistically significant. An interesting finding of this study, and our recent review on PET/SPECT imaging of EVs, 45 is the presence of sEV renal excretion that we have previously suggested may be related to small EV fragments from fast EV metabolism/decomposition, as sEVs are much larger than the ∼55 kDa renal filtration threshold. 46 Interestingly, the immunofluorescence microscopy data of the kidneys ( Figure 6C) strongly suggest the presence of human CD63 proteins in PANC1 sEV-treated mice, as a strong fluorescence signal can be observed in the tubules of intact PANC1 sEV-treated mice. This finding could be due to either whole PANC1 sEVs present in kidney tubules, which would agree with the higher amount of the 89 Zr signal from the biodistribution data, or CD63-containing fragments of sEVs that were able to pass through renal filtration.
For signal quantification, ROIs were drawn randomly to include areas of bright and weak fluorescence ( Figure 6D). Spleen fluorescence was significantly higher for intact sEVs compared to heat-damaged sEVs, corresponding to both PET imaging and ex vivo biodistribution. Moreover, both the heatdamaged sEV fluorescence and control group fluorescence show a similar low signal. This further reinforces the previous proposal ( Figure 5D) that the spleen uptake for 89 Zr-labeled PANC1 sEVs can be used as an imaging biomarker to determine the sEV's stability and quality. Correlating to in/ex vivo findings, there was no statistically significant difference between the intact and heat-damaged group for the liver and kidney. Although, according to the PET imaging and the biodistribution data, radioactivity detected in the liver is considerably higher than that detected in the kidneys, the fluorescence intensity level is very similar. As such, it can be proposed that once 89 Zr-labeled exosomes are taken up by the liver, any 89 Zr released from the vesicles is retained within this organ.
It is important to discuss the advantages and disadvantages of the radiolabeling method described in this report. Compared to other EV radiolabeling methods, 45 [ 89 Zr]Zr-(oxinate) 4 sEV radiolabeling benefits from radiochemical simplicity and low barriers for clinical translation, as this radiotracer is already being used in several preclinical and clinical trials for cell and liposomal nanomedicine tracking. The sEV RLY achieved is comparable to that reported for other sEV radiolabeling methods. Our data also strongly suggest that [ 89 Zr]Zr(oxinate) 4 sEV radiolabeling does not interfere with sEV membrane proteins, which is an advantage compared to methods that rely on covalent bond formation with membrane molecules (e.g., bifunctional chelator-based) and hence are more likely to bind and affect their structure/function. We note, however, that further studies (e.g., proteomics) would be required to fully validate this. We chose 89 Zr (t 1/2 = 3.3 d) due to its long half-life thus enabling PET tracking of sEVs for up to ca. >7 days. However, our in vitro stability studies showed ca. 25% release of 89 Zr from radiolabeled sEVs, and thus in vivo PET-CT imaging was limited to 24 h to avoid analysis errors due to excessive levels of released free 89 Zr. In terms of radiation dosimetry and potential clinical translation, indeed 89 Zr may not be the radionuclide of choice if imaging is limited within this timeframe. It is worth noting, however, that compared to other radiometals such as 64 Cu and 52 Mn, 89 Zr exhibits significantly better intravesicular/cellular retention. 31,47 PET-CT imaging of 89 Zr-PANC1 sEVs showed fast 89 Zr uptake in the liver, spleen, and brain and suspected accumulation in LNs, which was supported by immunofluorescence imaging. The imaging data and high human-CD63 signal in the kidneys support the hypothesis that some populations of sEVs and/or sEV fragments can be cleared renally. We have also demonstrated that heat-damaged 89 Zr-PANC1 sEVs show significant differences in spleen uptake, further supporting the key role this organ plays in the biodistribution of sEVs 48 and leading us to propose the spleen/ bone uptake ratio as an imaging biomarker for sEV stability when using [ 89 Zr]Zr(oxinate) 4 to radiolabel PANC1 sEVs.

■ CONCLUSIONS
We have developed and optimized the synthesis of [ 89 Zr]Zr-(oxinate) 4 and demonstrated that it allows simple, efficient, and direct labeling of sEVs. Using PANC1 sEVs as a model, our results demonstrated that sEVs retain their morphological characteristics following radiolabeling with [ 89 Zr]Zr(oxinate) 4 and also strongly suggest that surface biomolecules are not affected. In vivo PET-CT imaging in healthy mice showed that 89 Zr-labeled sEVs are stable for 24 h and thus can reliably be tracked within this timeframe. The differential spleen:bone uptake ratio for intact versus heat-damaged 89 Zr-PANC1 sEVs led to the proposition of using this parameter as an imaging biomarker for sEV stability when using this radiolabeling method. Further work will aim at understanding the nature of the extensive lymph node and brain 89 Zr uptake and using PET imaging to support the development of sEVs as nanotherapeutics. We believe that this radiochemical tool will help the field to further investigate the in vivo behavior of sEVs and answer questions on their basic biology, supporting their applications as delivery vehicles, disease biomarkers (e.g., identify metastatic niches), or as therapeutics.  4 and control 89 Zr (10−20 μL, 1 MBq) obtained by both formation methods were added to separate tubes, containing 500 μL of both PBS and octanol. Triplicate samples were prepared. The mixtures were vortexed at maximum speed for 3 min, followed by centrifugation at 16,000g for 3 min. Aliquots from each phase were transferred to separate Eppendorf tubes, and activities were measured using a gamma counter (Wallac Wizard 1282 CompuGamma, PerkinElmer).
Cell Culture. For sEV isolation, all cells were cultured in cell media supplemented by 10% exo-depleted foetal bovine serum (FBS). FBS was depleted of exosomes or sEVs by ultracentrifugation at 100,000g for 18 h at 4°C in a Beckman L60 ultracentrifuge with a SW41 Ti rotor (Beckman Coulter), followed by sterile filtration of the top two layers through a 0.22 μm PES membrane filter (Merck). MDA-MB-231.CD63-GFP, human metastatic breast cancer and PANC1, human metastatic pancreatic cancer cells were cultured in CELLine AD1000 bioreactor flasks (Wheaton) at 37°C and in 5% CO 2 , as described by Mitchell et al. 49 Cells were cultured in 15 mL of low glucose DMEM and RPMI 1640, respectively, supplemented with 10% exo-depleted FBS, 1% penicillin− streptomycin, and 1% L-glutamine (all supplied by Sigma-Aldrich) in the bottom cell chamber, with 500 mL of the same medium as before, except that exo-depleted FBS was replaced with standard FBS, in the top reservoir chamber of the bioreactor flask. The cell supernatant was collected weekly and replaced with fresh exo-depleted cell media. Medium in the reservoir chamber was also replaced weekly. Immediately after collection, the supernatant was subjected to centrifugation at 500g for 5 min twice followed by at 2000g for 15 min, then filtration through a 0.22 μm PES filter. This filtered conditioned medium (CM) was stored at 4°C for up to 6 weeks until used for sEV isolation.
sEV Isolation. MDA-MB-231.CD63-GFP and PANC1 sEVs were isolated by following a protocol described previously. 16 Briefly, 22.5 mL of CM was layered on 3 mL of 25% (w/w) sucrose cushion in D 2 O (Sigma-Aldrich) in a thick-walled polycarbonate centrifuge tube (Beckman Coulter) and ultracentrifuged (SW48 Ti rotor) at 100,000g for 1.5 h at 4°C . The sucrose layer was transferred to another thick-walled centrifuge tube containing PBS, followed by another ultracentrifugation step (70.1 Ti rotor) at 100,000g for 1.5 h at 4°C . Finally, the supernatant was discarded, and the sEV pellet was suspended in 200 μL of PBS and stored at 4°C.
Nanoparticle Tracking Analysis. The hydrodynamic diameter and concentration of sEVs were measured by NTA using NanoSight LM10, equipped with a 488 nm blue laser and NTA software v3.2 (Malvern Panalytical). The stock sample was diluted to achieve about 20−80 particles/viewing frame. Measurements were made in triplicates for 60 s, for up to three serial dilutions of the sample. Parameters used to capture and analyze data are as follows: screen gain = 2, camera level = 13, FPS = 25, viscosity = water, and detection threshold = 5.
Cryo-Electron Microscopy. QUANTIFOIL R 2/2 carbon grids (mesh: Cu 300, #234901; Agar Scientific) were plasma discharged for 50 s at 30 SCCM gas flow in Nanoclean 1070 (Fischione instruments). Aliquots (3 μL) of non-radiolabeled or 89 Zr-labeled PANC1 sEVs in PBS were deposited on the carbon grids in Vitrobot Mark IV (FEI). This was followed by blotting with standard Vitrobot filter paper (Agar Scientific) to remove excess liquid; blotting time = 2 s, wait time = 30 s, and blotting force = −2. The grids were then plunge frozen in liquid ethane (−188°C) and maintained in liquid N 2 (−196°C ) in a grid box and transferred into a cryo-transfer holder. CryoEM was performed on TECNAI 12 G 2 (FEI) connected to a TemCam-F216 camera and Temmenu v4 software (Tietz Video & Image Processing Systems GmbH, Germany). Parameters used to capture images are as follows: electron acceleration = 120 kV, magnification = 42,000×, acquisition time = 1 s, defocus = −2.5 to −3 μm, and spot size = 5. To minimize radiation damage during localization of sEVs, grids were visualized using the low-dose mode.
BCA Protein Assay. The protein content of the sEVs was analyzed in duplicates of up to three serial dilutions using Pierce Rapid Gold BCA protein assay (Thermo Fisher), according to the manufacturer's microplate protocol. Absorbance was measured at 480 nm on SPECTROstar Nano (BMG Labtech).
Radiolabeling of sEVs. MDA-MB-231.CD63-GFP sEVs, ca. 1 × 10 10 vesicles, and ca. 1 × 10 11 PANC1 sEVs in 160 μL of PBS were incubated with 20 μL of [ 89 Zr]Zr(oxinate) 4 or 89 Zr control for 20 min at 37°C with frequent shaking, followed by addition of 100 μL of 1% DFO (deferoxamine mesylate salt, ≥92.5%; Sigma) in PBS to trap any unbound 89 Zr. Radiolabeled sEVs were purified from an unchelated radiotracer by SEC using Exo-spin mini-HD columns (Cell Guidance Systems) or self-prepared Sepharose CL-2B resin (GE Healthcare). The resin was self-packed under gravity into empty G-25 MiniTrap columns (GE Healthcare). The reaction mixture was loaded onto the column, and the purified sample was eluted using the manufacturer's protocol for either mini-HD or minitrap columns. Radioactivity of the eluate and the column was measured using a gamma counter to calculate RLY.
Heat Damaging of 89 Zr-PANC1 sEVs. After radiolabeling, sEVs were damaged by a ×2 heat/cool cycle heating to 90°C for 20 min followed by incubation in ice for 10 min, repeated once more. Expression of sEV marker proteins after heat damage was analyzed by bead-assisted flow cytometry. To evaluate damage, sEVs were passed through an Exo-spin mini-HD column for characterization by NTA, BCA protein assay, and RLY.
In Vitro Stability of 89 Zr-PANC1 sEVs in PBS. 89 Zr-PANC1 sEVs (intact or heat-damaged) were incubated in PBS at 37°C for up to 72 h (n = 2 in duplicate for up to 24 h, n = 1 in duplicate thereafter). Stability was assessed by iTLC; stationary phase = Whatman No 1 paper (GE healthcare) and mobile phase = 10 mM EDTA at pH 6. 51 The chromatograms were analyzed on LabLogic Mini-Scan MS-1000F (Eckert & Ziegler) using a β detector probe and processed using Pearl software. In vitro stability was calculated by comparing the radioactivity associated at R f = 0 compared to the rest of the chromatogram.
PET-CT Imaging. Animal studies were carried out in accordance with the UK Home Office regulations under The Animals (Scientific Procedures) Act 1986. Immunocompetent C57BL/6j male mice (8−10 weeks) were anaesthetized with 2−2.5% isoflurane in 100% oxygen. 89  PET-CT imaging was performed on a nanoScan PET-CT preclinical imaging system (Mediso Medical Imaging System) using an air-heated standard single bed or a four-bed mouse hotel; 52 anesthesia was maintained throughout the scans. PET imaging was started at t = 0.5 h for 2 h and at t = 24 h for 1 h followed by a CT scan. All PET/CT data were reconstructed in Nucline v.0.21 (Mediso Medical Imaging System) using Monte Carlo-based Tera-Tomo 3D PET reconstruction (400− 600 keV energy window, 1−3 coincidence mode, and 4 iterations and 6 subsets) at an isotropic voxel size of 0.4 mm; images were corrected for scatter attenuation and were decay corrected to the time of injection. Reconstructed images were analyzed using VivoQuant (inviCRO Inc).
At the end of the imaging session at t = 24 h, mice were culled by cervical dislocation while under anesthesia. Blood, urine, and organs of interest were collected and weighed for the ex vivo biodistribution study. Standards of the injected radiotracer were prepared by serial dilutions. These standards along with the collected tissues were gamma counted to calculate the percentage injected dose (%ID/g).
Immunofluorescence Detection. Following in vivo imaging, the spleen, liver, and kidneys were fixed in 10% neutral buffered formalin at 4°C for up to 48 h, maintained in 70% ethanol until radioactivity decayed, and embedded in paraffin. Organ sections (5 μm) were de-paraffinized, and antigen retrieval was performed in 10 mM citrate buffer (pH 6) with 0.1% Tween-20 at 100°C for 20 min. Sections were blocked with 5% goat serum and 1% BSA for 1 h at RT and incubated in a rabbit anti-human CD63 (EPR5702, 1.9 μg/ mL; Abcam, # ab134045) antibody overnight at 4°C. Tissues were then stained with Cy5 (3 μg/mL; Jackson ImmunoResearch, #111-175-144) for 1 h at RT and mounted using Fluoroshield DAPI (Sigma). Confocal microscopy was performed on an Eclipse Ti-E A1 inverted confocal microscope with a Plan Apo λ 20× objective (Nikon), and images were analyzed on ImageJ. For signal quantification, images were split into separate channelsred and blue, and random ROIs were on the red channel grayscale image for Cy5 and quantified using the "analyze" and "measure" tool on ImageJ.
Bioconjugate Chemistry pubs.acs.org/bc Article Statistical Analysis. All numerical data were analyzed on GraphPad Prism 8 or Microsoft Excel 2016. All values are given in one decimal place. Data are presented as mean ± standard deviation (SD), unless stated otherwise. Unless specified, Student's unpaired t-test was used to calculate statistical differences between groups with the P value < 0.05 considered significant. Exact significance values are reported in each figure.

■ ACKNOWLEDGMENTS
The authors would like to thank Dr. Susanne Heck for her advice on flow cytometry data analysis, Dr. Tim Witney for a loan of a four-bed mouse hotel for preclinical PET/CT imaging, Dr. Oskar Timmermand and Aishwarya Mishra for assistance during dissection, and Dr. Ana Baburamani and Dr. Begonã Lavin-Plaza for guidance on immunohistochemistry techniques. We also thank the Nikon Imaging Centre at King's College London for use of their confocal microscope.